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1.  Inflammatory response and pneumocyte apoptosis during lung ischemia–reperfusion injury in an experimental pulmonary thromboembolism model 
Lung ischemia–reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. The inflammatory response mechanisms related to LIRI in pulmonary thromboembolism (PTE), especially in chronic PTE, need to be studied further. In a PTE model, inflammatory response and apoptosis may occur during LIRI and nitric oxide (NO) inhalation may alleviate the inflammatory response and apoptosis of pneumocytes during LIRI. A PTE canine model was established through blood clot embolism to the right lower lobar pulmonary artery. Two weeks later, we performed embolectomy with reperfusion to examine the LIRI changes among different groups. In particular, the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2), serum concentrations of tumor necrosis factor-α (TNF-α), myeloperoxidase concentrations in lung homogenates, alveolar polymorphonuclear neutrophils (PMNs), lobar lung wet to dry ratio (W/D ratio), apoptotic pneumocytes, and lung sample ultrastructure were assessed. The PaO2/FiO2 in the NO inhalation group increased significantly when compared with the reperfusion group 4 and 6 h after reperfusion (368.83 ± 55.29 vs. 287.90 ± 54.84 mmHg, P < 0.05 and 380.63 ± 56.83 vs. 292.83 ± 6 0.34 mmHg, P < 0.05, respectively). In the NO inhalation group, TNF-α concentrations and alveolar PMN infiltration were significantly decreased as compared with those of the reperfusion group, 6 h after reperfusion (7.28 ± 1.49 vs. 8.90 ± 1.43 pg/mL, P < 0.05 and [(19 ± 6)/10 high power field (HPF) vs. (31 ± 11)/10 HPF, P < 0.05, respectively]. The amount of apoptotic pneumocytes in the lower lobar lung was negatively correlated with the arterial blood PaO2/FiO2, presented a positive correlation trend with the W/D ratio of the lower lobar lung, and a positive correlation with alveolar PMN in the reperfusion group and NO inhalation group. NO provided at 20 ppm for 6 h significantly alleviated LIRI in the PTE model. Our data indicate that, during LIRI, an obvious inflammatory response and apoptosis occur in our PTE model and NO inhalation may be useful in treating LIRI by alleviating the inflammatory response and pneumocyte apoptosis. This potential application warrants further investigation.
PMCID: PMC4445764  PMID: 25677043
Lung ischemia–reperfusion injury; Nitric oxide inhalation; Pulmonary thromboembolism; Inflammatory response; Pneumocyte apoptosis
2.  Beneficial effects of inhaled NO on apoptotic pneumocytes in pulmonary thromboembolism model 
Lung ischemia–reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. Inhaled NO may be useful in treating acute and chronic pulmonary thromboembolism (PTE) due to the biological effect property of NO.
A PTE canine model was established through selectively embolizing blood clots to an intended right lower lobar pulmonary artery. PaO2/FiO2, the mPAP and PVR were investigated at the time points of 2, 4, 6 hours after inhaled NO. Masson’s trichrome stain, apoptotic pneumocytes and lung sample ultrastructure were also investigated among different groups.
The PaO2/FiO2 in the Inhaled NO group increased significantly when compared with the Reperfusion group at time points of 4 and 6 hours after reperfusion, mPAP decreased significantly at point of 2 hours and the PVR decreased significantly at point of 6 hours after reperfusion. The amounts of apoptotic type II pneumocytes in the lower lobar lung have negative correlation trend with the arterial blood PaO2/FiO2 in Reperfusion group and Inhaled NO group. Inhaled nitric oxide given at 20 ppm for 6 hours can significantly alleviate the LIRI in the model.
Dramatic physiological improvements are seen during the therapeutic use of inhaled NO in pulmonary thromboembolism canine model. Inhaled NO may be useful in treating LIRI in acute or chronic PTE by alleviating apoptotic type II pneumocytes. This potential application warrants further investigation.
PMCID: PMC4135342  PMID: 25109474
Lung ischemia-reperfusion injury; Pulmonary thromboembolism; Apoptotic pneumocytes; Inhaled nitric oxide
3.  RC-3095, a Selective Gastrin-Releasing Peptide Receptor Antagonist, Does Not Protect the Lungs in an Experimental Model of Lung Ischemia-Reperfusion Injury 
BioMed Research International  2015;2015:496378.
RC-3095, a selective GRPR antagonist, has been shown to have anti-inflammatory properties in different models of inflammation. However, its protective effect on lungs submitted to lung ischemia-reperfusion injury has not been addressed before. Then, we administrated RC-3095 intravenously before and after lung reperfusion using an animal model of lung ischemia-reperfusion injury (LIRI) by clamping the pulmonary hilum. Twenty Wistar rats were subjected to an experimental model in four groups: SHAM, ischemia-reperfusion (IR), RC-Pre, and RC-Post. The final mean arterial pressure significantly decreased in IR and RC-Pre compared to their values before reperfusion (P < 0.001). The RC-Post group showed significant decrease of partial pressure of arterial oxygen at the end of the observation when compared to baseline (P = 0.005). Caspase-9 activity was significantly higher in the RC-Post as compared to the other groups (P < 0.013). No significant differences were observed in eNOS activity among the groups. The groups RC-Pre and RC-Post did not show any significant decrease in IL-1β (P = 0.159) and TNF-α (P = 0.260), as compared to IR. The histological score showed no significant differences among the groups. In conclusion, RC-3095 does not demonstrate a protective effect in our LIRI model. Additionally, its use after reperfusion seems to potentiate cell damage, stimulating apoptosis.
PMCID: PMC4393930  PMID: 25893195
4.  Influence of Levosimendan Postconditioning on Apoptosis of Rat Lung Cells in a Model of Ischemia–Reperfusion Injury 
PLoS ONE  2015;10(1):e0114963.
To ascertain if levosimendan postconditioning can alleviate lung ischemia–reperfusion injury (LIRI) in rats.
One hundred rats were divided into five groups: Sham (sham), ischemia–reperfusion group (I/R group), ischemic postconditioning (IPO group), levosimendan postconditioning (Levo group) and combination postconditioning group of levosimendan and 5-Hydroxydecanoic acid (Levo+5-HD group). The apoptotic index (AI) of lung tissue cells was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Expression of active cysteine aspartate specific protease-3 ( active caspase-3), Bcl-2 and Bax in lung tissue was determined by immunohistochemical staining. The morphopathology of lung tissue was observed using light and electron microscopy.
AI values and expression of active caspase-3, Bcl-2 and Bax of lung tissue in I/R and Levo+5-HD groups were significantly higher than those in the sham group ( P<0.05). AI values and expression of active caspase-3 and Bax were significantly lower, whereas that of Bcl-2 was higher significantly in the Levo group, compared with I/R and Levo+5-HD groups (P<0.05). Significant differences were not observed in comparisons between I/R and Levo+5-HD groups as well as IPO and Levo groups.
LIRI can be alleviated by levosimendan, which simulates an IPO protective function. A postulated lung-protective mechanism of action could involve opening of mitochondrial adenosine triphosphate-sensitive potassium channels, relieving Ca2+ overload, upregulation of expression of Bcl-2, and downregulation of expression of active caspase-3 and Bax.
PMCID: PMC4301642  PMID: 25608001
5.  Mesenchymal stem cell pretreatment of non-heart-beating-donors in experimental lung transplantation 
Lung transplantation (LTx) is still limited by organ shortage. To expand the donor pool, lung retrieval from non-heart-beating donors (NHBD) was introduced into clinical practice recently. However, primary graft dysfunction with inactivation of endogenous surfactant due to ischemia/reperfusion-injury is a major cause of early mortality. Furthermore, donor-derived human mesenchymal stem cell (hMSC) expansion and fibrotic differentiation in the allograft results in bronchiolitis obliterans syndrome (BOS), a leading cause of post-LTx long-term mortality. Therefore, pretreatment of NHBD with recipient-specific bone-marrow-(BM)-derived hMSC might have the potential to both improve the postischemic allograft function and influence the long-term development of BOS by the numerous paracrine, immunomodulating and tissue-remodeling properties especially on type-II-pneumocytes of hMSC.
Asystolic pigs (n = 5/group) were ventilated for 3 h of warm ischemia (groups 2–4). 50x106 mesenchymal-stem-cells (MSC) were administered in the pulmonary artery (group 3) or nebulized endobronchially (group 4) before lung preservation. Following left-lung-transplantation, grafts were reperfused, pulmonary-vascular-resistance (PVR), oxygenation and dynamic-lung-compliance (DLC) were monitored and compared to control-lungs (group 2) and sham-controls (group 1). To prove and localize hMSC in the lung, cryosections were counter-stained. Intra-alveolar edema was determined stereologically. Statistics comprised ANOVA with repeated measurements.
Oxygenation (p = 0.001) and PVR (p = 0.009) following endovascular application of hMSC were significantly inferior compared to Sham controls, whereas DLC was significantly higher in endobronchially pretreated lungs (p = 0.045) with overall sham-comparable outcome regarding oxygenation and PVR. Stereology revealed low intrapulmonary edema in all groups (p > 0.05). In cryosections of both unreperfused and reperfused grafts, hMSC were localized in vessels of alveolar septa (endovascular application) and alveolar lumen (endobronchial application), respectively.
Preischemic deposition of hMSC in donor lungs is feasible and effective, and endobronchial application is associated with significantly better DLC as compared to sham controls. In contrast, transvascular hMSC delivery results in inferior oxygenation and PVR. In the long term perspective, due to immunomodulatory, paracrine and tissue-remodeling effects on epithelial and endothelial restitution, an endobronchial NHBD allograft-pretreatment with autologous mesenchymal-stem-cells to attenuate limiting bronchiolitis-obliterans-syndrome in the long-term perspective might be promising in clinical lung transplantation. Subsequent work with chronic experiments is initiated to further elucidate this important field.
PMCID: PMC4169637  PMID: 25179441
Non-heart-beating donors; Perfadex lung preservation; Mesenchymal stem cell therapy; Ischemia-reperfusion injury; Donor pretreatment
6.  Protective Role of Matrix Metalloproteinase-9 in Ozone-Induced Airway Inflammation 
Environmental Health Perspectives  2007;115(11):1557-1563.
Exposure to ozone causes airway inflammation, hyperreactivity, lung hyper-permeability, and epithelial cell injury. An early inflammatory response induced by inhaled O3 is characterized primarily by release of inflammatory mediators such as cytokines, chemokines, and airway neutrophil accumulation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of oxidative lung disorders including acute lung injury, asthma, and chronic obstructive pulmonary disease.
We hypothesized that MMPs have an important role in the pathogenesis of O3-induced airway inflammation.
We compared the lung injury responses in either Mmp7- (Mmp7−/−) or Mmp9-deficient (Mmp9−/−) mice and their wild-type controls (Mmp7+/+, Mmp9+/+) after exposure to 0.3 ppm O3 or filtered air.
Relative to air-exposed controls, MMP-9 activity in bronchoalveolar lavage fluid (BALF) was significantly increased by O3 exposure in Mmp9+/+ mice. O3-induced increases in the concentration of total protein (a marker of lung permeability) and the numbers of neutrophils and epithelial cells in BALF were significantly greater in Mmp9−/− mice compared with Mmp9+/+ mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 levels in BALF were also significantly higher in Mmp9−/− mice than in Mmp9+/+ mice after O3 exposure, although no differences in mRNA expression for these chemokines were found between genotypes. Mean BALF protein concentration and numbers of inflammatory cells were not significantly different between Mmp7+/+ and Mmp7−/− mice after O3 exposure.
Results demonstrated a protective role of MMP-9 but not of MMP-7, in O3-induced lung neutrophilic inflammation and hyperpermeability. The mechanism through which Mmp9 limits O3-induced airway injury is not known but may be via posttranscriptional effects on proinflammatory CXC chemokines including KC and MIP-2.
PMCID: PMC2072825  PMID: 18007984
chemokine; knockout mice; lung; MMP-9; O3; oxidant
7.  Exogenous surfactant application in a rat lung ischemia reperfusion injury model: effects on edema formation and alveolar type II cells 
Respiratory Research  2008;9(1):5.
Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells.
Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4°C and 50 min of reperfusion at 37°C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells.
Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 μm3(0.10)) and CE+S (481 μm3(0.10)) compared with controls (323 μm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05).
Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant.
PMCID: PMC2265285  PMID: 18205928
8.  Prevention of LPS-Induced Acute Lung Injury in Mice by Mesenchymal Stem Cells Overexpressing Angiopoietin 1 
PLoS Medicine  2007;4(9):e269.
The acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar–capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Current specific treatment strategies for ALI/ARDS are lacking. We hypothesized that mesenchymal stem cells (MSCs), with or without transfection with the vasculoprotective gene angiopoietin 1 (ANGPT1) would have beneficial effects in experimental ALI in mice.
Methods and Findings
Syngeneic MSCs with or without transfection with plasmid containing the human ANGPT1 gene (pANGPT1) were delivered through the right jugular vein of mice 30 min after intratracheal instillation of lipopolysaccharide (LPS) to induce lung injury. Administration of MSCs significantly reduced LPS-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts in bronchoalveolar lavage (BAL) fluid (53%, 95% confidence interval [CI] 7%–101%; and 60%, CI 4%–116%, respectively) as well as reducing levels of proinflammatory cytokines in both BAL fluid and lung parenchymal homogenates. Furthermore, administration of MSCs transfected with pANGPT1 resulted in nearly complete reversal of LPS-induced increases in lung permeability as assessed by reductions in IgM and albumin levels in BAL (96%, CI 6%–185%; and 74%, CI 23%–126%, respectively). Fluorescently tagged MSCs were detected in the lung tissues by confocal microscopy and flow cytometry in both naïve and LPS-injured animals up to 3 d.
Treatment with MSCs alone significantly reduced LPS-induced acute pulmonary inflammation in mice, while administration of pANGPT1-transfected MSCs resulted in a further improvement in both alveolar inflammation and permeability. These results suggest a potential role for cell-based ANGPT1 gene therapy to treat clinical ALI/ARDS.
Using a mouse model of acute respiratory distress syndrome, Duncan Stewart and colleagues report that rescue with mesenchymal stem cells expressing human angiopoietin 1 can avert lung injury from lipopolysaccharide.
Editors' Summary
Critically ill people who have had an injury to their lungs, for example through pneumonia, trauma, or an immune response to infection, may end up developing a serious complication in the lung termed acute respiratory distress syndrome (ARDS). In ARDS, inflammation develops in the lung, and fluid builds up in the alveoli (the air sacs resembling “bunches of grapes” at the ends of the network of tubes in the lung). This buildup of fluid prevents oxygen from being carried efficiently from air into the blood; the individual consequently experiences problems breathing and can develop further serious complications, which contribute significantly to the burden of illness among people in intensive care units. The death rate among individuals who do develop ARDS is very high, upward of 30%. Normally, individuals with ARDS are given extra oxygen, and may need a machine to help them breathe; treatments also focus on addressing the underlying causes in each particular patient. However, currently there are very few specific treatments that address ARDS itself.
Why Was This Study Done?
The researchers here wanted to work toward new treatment options for individuals with ARDS. One possible approach involves cells known as mesenchymal stem cells (MSCs). These cells are typically found in the bone marrow and have a property shared by very few other cell types in the body; they are able to carry on dividing and renewing themselves, and can eventually develop into many other types of cell. The researchers already knew that MSCs could become incorporated into injured lungs in mice and develop there into the tissue layers lining the lung. Some interesting work had also been done on a protein called angiopoeitin 1 (ANGPT1), which seemed to play a role in protecting against inflammation in blood vessels. Therefore, there was a strong rationale for carrying out experiments in mice to see if MSCs engineered to produce the ANGPT1 protein might “rescue” lung injury in mice. These experiments would be an initial step toward developing possible new treatments for humans with ARDS.
What Did the Researchers Do and Find?
The researchers used a mouse model to mimic the human ARDS condition. This involved injecting the windpipe of experimental mice with lipopolysaccharide (a substance normally found on the outer surface of bacteria that brings about an immune reaction in the lung). After 30 minutes, the mice were then injected with either salt solution (as a control), the MSCs, or MSCs producing the ANGPT1 protein. The researchers then looked at markers of lung inflammation, the appearance of the lungs under a microscope, and whether the injected MSCs had become incorporated into the lung tissue.
The lipopolysaccharide brought about a large increase in the number of inflammatory cells in the lung fluid, which was reduced in the mice given MSCs. Furthermore, in mice given the MSCs producing ANGPT1 protein, the number of inflammatory cells was reduced to a level similar to that of mice that had not been given lipopolysaccharide. When the researchers looked at the appearance under the microscope of lungs from mice that had been given lipopolysaccharide, they saw signs of inflammation and fluid coming out into the lung air spaces. These signs were reduced among both mice treated with MSCs and those treated with MSCs producing ANGPT1. The researchers also measured the “leakiness” of the lung tissues in lipopolysaccharide-treated mice; MSCs seemed to reduce the leakiness to some extent, and the lungs of mice treated with MSCs producing ANGPT1 were no more leaky than those of mice that had never been injected with lipopolysaccharide. Finally, the MSCs were seen to be incorporated into lung tissue by three days after injection, but after that were lost from the lung.
What Do These Findings Mean?
Previous research done by the same group had shown that fibroblasts producing ANGPT1 could prevent lung injury in rats later given lipopolysaccharide. The experiments reported here go a step further than this, and suggest that MSCs producing ANGPT1 can “rescue” the condition of mouse lungs that had already been given lipopolysaccharide. In addition, treatment with MSCs alone also produced beneficial effects. This opens up a possible new treatment strategy for ARDS in humans. However, it should be emphasized that the animal model used here is not a precise parallel of ARDS in humans, and that more research remains to be done before human studies of this sort could be considered.
Additional Information.
Please access these Web sites via the online version of this summary at
Medline Plus entry on acute respiratory distress syndrome, providing basic information about what ARDS is, its effects, and how it is currently managed
ARDS Network from the US National Heart, Lung, and Blood Institute of the National Institutes of Health; the site provides frequently asked questions about ARDS as well as a list of clinical trials conducted by the network
Information about stem cells from the US National Institutes of Health, including information about the potential uses of stem cells
Wikipedia page about mesenchymal stem cells (note: Wikipedia is an internet encyclopedia anyone can edit)
PMCID: PMC1961632  PMID: 17803352
9.  Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice 
Respiratory Research  2009;10(1):79.
To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.
PMCID: PMC2751757  PMID: 19706153
10.  Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair 
Respiratory Research  2009;10(1):116.
Preterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury in vitro, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised.
BALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (≥ 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model.
On day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 ± 35.5 vs. -6.3 ± 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 ± 87.1 vs. 40.2 ± 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1.
Antenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair in vitro. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.
PMCID: PMC2784455  PMID: 19930634
11.  Protection from pulmonary ischemia-reperfusion injury by adenosine A2A receptor activation 
Respiratory Research  2009;10(1):58.
Lung ischemia-reperfusion (IR) injury leads to significant morbidity and mortality which remains a major obstacle after lung transplantation. However, the role of various subset(s) of lung cell populations in the pathogenesis of lung IR injury and the mechanisms of cellular protection remain to be elucidated. In the present study, we investigated the effects of adenosine A2A receptor (A2AAR) activation on resident lung cells after IR injury using an isolated, buffer-perfused murine lung model.
To assess the protective effects of A2AAR activation, three groups of C57BL/6J mice were studied: a sham group (perfused for 2 hr with no ischemia), an IR group (1 hr ischemia + 1 hr reperfusion) and an IR+ATL313 group where ATL313, a specific A2AAR agonist, was included in the reperfusion buffer after ischemia. Lung injury parameters and pulmonary function studies were also performed after IR injury in A2AAR knockout mice, with or without ATL313 pretreatment. Lung function was assessed using a buffer-perfused isolated lung system. Lung injury was measured by assessing lung edema, vascular permeability, cytokine/chemokine activation and myeloperoxidase levels in the bronchoalveolar fluid.
After IR, lungs from C57BL/6J wild-type mice displayed significant dysfunction (increased airway resistance, pulmonary artery pressure and decreased pulmonary compliance) and significant injury (increased vascular permeability and edema). Lung injury and dysfunction after IR were significantly attenuated by ATL313 treatment. Significant induction of TNF-α, KC (CXCL1), MIP-2 (CXCL2) and RANTES (CCL5) occurred after IR which was also attenuated by ATL313 treatment. Lungs from A2AAR knockout mice also displayed significant dysfunction, injury and cytokine/chemokine production after IR, but ATL313 had no effect in these mice.
Specific activation of A2AARs provides potent protection against lung IR injury via attenuation of inflammation. This protection occurs in the absence of circulating blood thereby indicating a protective role of A2AAR activation on resident lung cells such as alveolar macrophages. Specific A2AAR activation may be a promising therapeutic target for the prevention or treatment of pulmonary graft dysfunction in transplant patients.
PMCID: PMC2711962  PMID: 19558673
12.  Matrix Metalloproteinase-8 Inactivates Macrophage Inflammatory Protein-1α to Reduce Acute Lung Inflammation and Injury in Mice 
To determine the role of matrix metalloproteinase-8 (MMP-8) in acute lung injury (ALI), we delivered LPS or bleomycin by the intratracheal route to MMP-8−/− mice versus WT mice or subjected the mice to hyperoxia (95% O2) and measured lung inflammation and injury at intervals. MMP-8−/− mice with ALI had greater increases in lung PMN and macrophage counts, measures of alveolar capillary barrier injury, lung elastance, and mortality than WT mice with ALI. Bronchoalveolar lavage fluid (BALF) from LPS-treated MMP-8−/− mice had more macrophage inflammatory protein-1α (MIP-1α) than BALF from LPS-treated WT mice, but similar levels of other pro- and anti-inflammatory mediators. MIP-1α−/− mice with ALI had less acute lung inflammation and injury than WT mice with ALI, confirming that MIP-1α promotes acute lung inflammation and injury in mice. Genetically deleting MIP-1α in MMP-8−/− mice abrogated the increased lung inflammation and injury and mortality in MMP-8−/− mice with ALI. Soluble MMP-8 cleaved and inactivated MIP-1α in vitro, but membrane-bound MMP-8 on activated PMNs had greater MIP-1α-degrading activity than soluble MMP-8. High levels of membrane-bound MMP-8 were detected on lung PMNs from LPS-treated WT mice, but soluble, active MMP-8 was not detected in BALF samples. Thus, MMP-8 has novel roles in restraining lung inflammation and in limiting alveolar capillary barrier injury during ALI in mice by inactivating MIP-1α. In addition, membrane-bound MMP-8 on activated lung PMNs is likely to be the key bioactive form of the enzyme that limits lung inflammation and alveolar capillary barrier injury during ALI.
PMCID: PMC2938777  PMID: 20042585
rodent; neutrophil; inflammation; knockout mice; chemokine
13.  An alternative therapy for idiopathic pulmonary fibrosis by doxycycline through matrix metalloproteinase inhibition 
Idiopatiic pulmonary fibrosis (IPF) is a disease of dysregulated fibrogenesis with abnormal matrix metalloproteinase (MMPs) activity, angiogenesis, and profibrotic milieu wherein MMPs inhibition appears to be target-based therapy. We evaluated the role of doxycycline as a nonspecific inhibitor of MMPs in IPF patients.
Materials and Methods
Patients of IPF diagnosed on the basis of ATS-ERS consensus criteria were put on oral doxycycline in an open prospective trial. They were followed up for long term with spirometry, 6 min walk test (6MWT), St. Georges respiratory questionnaire (SGRQ), forced vital capacity (FVC), and repeat bronchoscopy while on doxycycline monotherapy for over 24 weeks. Both the initial and follow-up broncho alveolar lavage fluids (BALF) from IPF patients (n = 6) and control subjects (n = 6) were looked for MMP-9, -3, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) expression. Additionally, doxycycline's action on MMP activities in vitro was tested in BALF of IPF patients.
Doxycycline intervention showed significant improvement in IPF patients in terms of change in 6MWT, SGRQ, FVC, and quality of life. The level of MMP-9, -3, TIMP-1 and VEGF in the BALF were found significantly higher in the IPF patients compared to the controls while doxycycline therapy reduced those parameters nearer to control value. Doxycycline also showed a significant dose-dependent reduction in the in vitro MMPs activities in BALF.
Doxycycline shows significant prospect in the treatment of IPF through its anti MMPs activities. This is the first report on a case series of long-term doxycycline monotherapy in IPF patients.
PMCID: PMC3162753  PMID: 21886950
Broncho alveolar lavage fluid; doxycycline; idiopathic pulmonary fibrosis; matrix metalloproteinase; vascular endothelial growth factor
14.  Metformin attenuates ventilator-induced lung injury 
Critical Care  2012;16(4):R134.
Diabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury.
Twenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF.
High-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation.
Pretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.
PMCID: PMC3580719  PMID: 22827994
15.  Characterization of lung stem cell niches in a mouse model of bleomycin-induced fibrosis 
In lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue-resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process.
Short term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and brochoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU+ and BrdU- cells were characterized by hematopoietic (CD45+), pluripotency (TTF1+, Oct3/4+, SSEA-3+, SSEA-4+, Sca1+, Lin-, CD34+, CD31+), and lung lineage-specific (SPC+, AQP-5+, CC-10+) markers. Clonogenic potential of LRCs were measured by CFU-c assays.
STA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21.LTA- Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU+ (LRC) were the putative stem cells. BrdU+CD45+ cells increased by 0.7-fold and SPC+CC10+ bronchoalveolar stem cells (BASC), decreased by ~40-fold post-bleomycin. BrdU+VEGF+ cells decreased by 1.8-fold while BrdU-VEGF+ cells increased 4.6-fold. Most BrdU- cells were CD45-. BrdU- BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU+ cells remained similar in control and bleomycin-treated lungs.
STA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/- but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) - significance is unknown. LTA- The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF+ LRC may indicate a signal for progenitor mobilization from niches. BrdU- BASC population may be a small quiescent population that remains as a reserve for more severe lung injury. Increase in VEGF+ non-LRC may indicate a checkpoint to counterbalance the mobilization of VEGF+ cells from the stem cell niche.
PMCID: PMC3392768  PMID: 22643035
16.  Protective Effect of Surfactant Inhalation against Warm Ischemic Injury in an Isolated Rat Lung Ventilation Model 
PLoS ONE  2013;8(8):e72574.
Warm ischemia-reperfusion injury remains a crucial issue in transplantation following the cardiac death of donors. Previously, we showed that surfactant inhalation during warm ischemia mitigated ischemia-reperfusion injury. This study investigated the mechanisms of surfactant inhalation protection of the warm ischemic lung after reoxygenation with ventilation alone. In an isolated rat lung ventilation model, cardiac arrest was induced in the CTRL (control) and SURF (surfactant treatment) groups by ventricular fibrillation. Ventilation was restarted 110 min later; the lungs were flushed, and a heart and lung block was procured. In the SURF group, a natural bovine surfactant (Surfacten®) was inhaled for 3 min at the end of warm ischemia. In the Sham (no ischemia) group, lungs were flushed, procured, and ventilated in the same way. Afterwards, the lungs were ventilated with room air without reperfusion for 60 min. Surfactant inhalation significantly improved dynamic compliance and airway resistance. Moreover, surfactant inhalation significantly decreased inducible nitric oxide synthase and caspase-3 transcript levels, and increased those of Bcl-2 and surfactant protein-C. Immunohistochemically, lungs in the SURF group showed weaker staining for 8-hydroxy-2′-deoxyguanosine, inducible nitric oxide synthase, and apoptosis, and stronger staining for Bcl-2 and surfactant protein-C. Our results indicate that surfactant inhalation in the last phase of warm ischemia mitigated the injury resulting from reoxygenation after warm ischemia. The reduction in oxidative damage and the inhibition of apoptosis might contribute to the protection of the warm ischemic lungs.
PMCID: PMC3757025  PMID: 24009692
17.  Improved lung preservation relates to an increase in tubular myelin-associated surfactant protein A 
Respiratory Research  2005;6(1):60.
Declining levels of surfactant protein A (SP-A) after lung transplantation are suggested to indicate progression of ischemia/reperfusion (IR) injury. We hypothesized that the previously described preservation-dependent improvement of alveolar surfactant integrity after IR was associated with alterations in intraalveolar SP-A levels.
Using immuno electron microscopy and design-based stereology, amount and distribution of SP-A, and of intracellular surfactant phospholipids (lamellar bodies) as well as infiltration by polymorphonuclear leukocytes (PMNs) and alveolar macrophages were evaluated in rat lungs after IR and preservation with EuroCollins or Celsior.
After IR, labelling of tubular myelin for intraalveolar SP-A was significantly increased. In lungs preserved with EuroCollins, the total amount of intracellular surfactant phospholipid was reduced, and infiltration by PMNs and alveolar macrophages was significantly increased. With Celsior no changes in infiltration or intracellular surfactant phospholipid amount occurred. Here, an increase in the number of lamellar bodies per cell was associated with a shift towards smaller lamellar bodies. This accounts for preservation-dependent changes in the balance between surfactant phospholipid secretion and synthesis as well as in inflammatory cell infiltration.
We suggest that enhanced release of surfactant phospholipids and SP-A represents an early protective response that compensates in part for the inactivation of intraalveolar surfactant in the early phase of IR injury. This beneficial effect can be supported by adequate lung preservation, as e.g. with Celsior, maintaining surfactant integrity and reducing inflammation, either directly (via antioxidants) or indirectly (via improved surfactant integrity).
PMCID: PMC1187923  PMID: 15969762
18.  Plasma Receptor for Advanced Glycation Endproducts Predicts Duration of ICU Stay and Mechanical Ventilation in Patients Following Lung Transplantation 
Primary graft dysfunction, formerly termed reperfusion pulmonary edema, is the leading cause of short-term complications after lung transplantation. New evidence shows that alveolar type I epithelial cells play an active role in alveolar fluid transport and are therefore presumed to be critical in the absorption of pulmonary edema. We tested the potential relevance of a novel marker of alveolar type I cell injury, the receptor for advanced glycation end products (RAGE), to short-term outcomes of lung transplantation.
Prospective, observational cohort study of 20 patients undergoing single lung, bilateral lung, or combined heart-lung transplantation. Plasma biomarkers were measured 4 hours after allograft reperfusion.
Higher plasma RAGE levels were associated with both a longer duration of mechanical ventilation and longer intensive care unit length of stay, in contrast to markers of alveolar type II cell injury, endothelial injury, and acute inflammation. Specifically, for every doubling in plasma RAGE levels, the duration of mechanical ventilation increased on average by 26 hours, adjusting for ischemia time (95% CI 7.4-44.7 hours, p=0.01). Likewise, for every doubling of plasma RAGE levels, intensive care unit length of stay increased on average by 1.8 days, again adjusting for ischemia time (95% CI 0.13-3.45; p=0.04). In contrast, the clinical diagnosis of primary graft dysfunction was not predictive of these short-term outcomes.
Higher levels of plasma RAGE measured shortly after reperfusion predicted poor short-term outcomes from lung transplantation. Elevated plasma RAGE levels may have both pathogenetic and prognostic value in patients following lung transplantation.
PMCID: PMC2741136  PMID: 17613396
Primary graft dysfunction; reperfusion pulmonary edema; biomarkers; alveolar epithelium; acute lung injury
19.  Acute phase proteins as local biomarkers of respiratory infection in calves 
BMC Veterinary Research  2015;11:167.
Cumulating reports suggest that acute phase proteins (APPs) do not only play a role as systemic inflammatory mediators, but are also expressed in different tissues as local reaction to inflammatory stimuli. The present study aimed to evaluate presence and changes in luminal lung concentrations of the APPs haptoglobin (Hp), lipopolysaccharide binding protein (LBP), C-reactive protein (CRP), and lactoferrin (Lf) in calves with an acute respiratory disease experimentally induced by Chlamydia (C.) psittaci.
Intra-bronchial inoculation of the pathogen resulted in a consistent respiratory illness. In venous blood of the infected calves (n = 13), concentrations of plasma proteins and serum LBP were assessed (i) before exposure and (ii) 8 times within 14 days after inoculation (dpi). Increasing clinical illness correlated significantly with increasing LBP—and decreasing albumin concentrations in blood, both verifying a systemic acute phase response.
Broncho-alveolar lavage fluid (BALF) was obtained from all 13 calves experimentally infected with C. psittaci at 4, 9 and 14 dpi, and from 6 uninfected healthy calves. Concentrations of bovine serum albumin (BSA), Hp, LBP, CRP and Lf in BALF were determined by ELISA. In infected animals, absolute concentrations of LBP and Hp in BALF correlated significantly with the respiratory score. The quotient [LBP]/[BSA] in BALF peaked significantly in acutely infected animals (4 dpi), showed a time-dependent decrease during the recovery phase (9-14 dpi), and was significantly higher compared to healthy controls. Concentrations of Hp and Lf in BALF as well as [Hp]/[BSA]—and [Lf]/[BSA]-quotients decreased during the study in infected animals, but were never higher than in healthy controls. CRP concentrations and [CRP]/[BSA]-quotient did not express significant differences between infected and healthy animals or during the course of infection.
In conclusion, absolute concentrations of LBP in blood and BALF as well as the quotient [LBP]/[BSA] in BALF perfectly paralleled the clinical course of respiratory illness after infection. Beside LBP, the suitability of Hp and Lf as local biomarkers of respiratory infections in cattle and their role in the local response to pathogens is worth further investigation, while CRP does not seem to play a role in local defense mechanisms of the bovine lung.
PMCID: PMC4515006  PMID: 26209015
Chlamydia psittaci; Acute phase proteins; Lung; Blood; Broncho-alveolar lavage; Bovine; Respiratory infection
20.  Prednisolone as Preservation Additive Prevents from Ischemia Reperfusion Injury in a Rat Model of Orthotopic Lung Transplantation 
PLoS ONE  2013;8(8):e73298.
The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect.
PMCID: PMC3756949  PMID: 24009745
21.  Essential Role of MMP-12 in Fas-Induced Lung Fibrosis 
Acute lung injury (ALI) is characterized by an early inflammatory response followed by a late fibroproliferative phase, and by an increase in the bronchoalveolar lavage fluid (BALF) concentrations of bioactive soluble FasL (sFasL). Activation of Fas (CD95) has been associated with the development of lung fibrosis in mice. The goal of this study was to determine the mechanisms that link Fas activation with the development of fibrosis in the lungs. We treated mice with three daily intratracheal instillations of a Fas-activating monoclonal antibody (Jo2) or a control IgG, and studied the animals at sequential times. Mice treated with Jo2 had increased caspase-3 activation in alveolar wall cells on Days 2, 4, and 7; an inflammatory response peaking on Day 7, and increased total lung collagen on Day 21. Gene expression profiling performed on Days 2, 4, and 7 showed sequential activation of co-regulated profibrotic genes, including marked up-regulation of matrix metalloproteinase 12 (MMP-12). Targeted deletion of MMP-12 protected mice from Fas-induced pulmonary fibrosis, even though the inflammatory responses in the lungs were similar to those of wild-type mice. Compared with wild-type mice, the mmp12−/− mice showed decreased expression of the profibrotic genes egr1 and cyr61. We conclude that Fas activation in the lungs induces a complex response that includes apoptosis, inflammation, and eventually fibrosis, and that MMP-12 is essential for the fibrotic phenotype. We speculate that MMP-12 activity is required for activation of the profibrotic genes egr1 and cyr61.
PMCID: PMC1976544  PMID: 17446527
apoptosis; inflammation; MMP-12; Fas; CYR61/CCN1
22.  A flavanone from Baccharis retusa (Asteraceae) prevents elastase-induced emphysema in mice by regulating NF-κB, oxidative stress and metalloproteinases 
Respiratory Research  2015;16(1):79.
Pulmonary emphysema is characterized by irreversible airflow obstruction, inflammation, oxidative stress imbalance and lung remodeling, resulting in reduced lung function and a lower quality of life. Flavonoids are plant compounds with potential anti-inflammatory and antioxidant effects that have been used in folk medicine. Our aim was to determine whether treatment with sakuranetin, a flavonoid extracted from the aerial parts of Baccharis retusa, interferes with the development of lung emphysema.
Intranasal saline or elastase was administered to mice; the animals were then treated with sakuranetin or vehicle 2 h later and again on days 7, 14 and 28. We evaluated lung function and the inflammatory profile in bronchoalveolar lavage fluid (BALF). The lungs were removed to evaluate alveolar enlargement, extracellular matrix fibers and the expression of MMP-9, MMP-12, TIMP-1, 8-iso-PGF-2α and p65-NF-κB in the fixed tissues as well as to evaluate cytokine levels and p65-NF-κB protein expression.
In the elastase-treated animals, sakuranetin treatment reduced the alveolar enlargement, collagen and elastic fiber deposition and the number of MMP-9- and MMP-12-positive cells but increased TIMP-1 expression. In addition, sakuranetin treatment decreased the inflammation and the levels of TNF-α, IL-1β and M-CSF in the BALF as well as the levels of NF-κB and 8-iso-PGF-2α in the lungs of the elastase-treated animals. However, this treatment did not affect the changes in lung function.
These data emphasize the importance of oxidative stress and metalloproteinase imbalance in the development of emphysema and suggest that sakuranetin is a potent candidate that should be further investigated as an emphysema treatment. This compound may be useful for counteracting lung remodeling and oxidative stress and thus attenuating the development of emphysema.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-015-0233-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4489216  PMID: 26122092
Emphysema; Flavonoid; NF-κB; Oxidative stress; Lung remodeling; Experimental models
23.  Lung inflammation and lack of genotoxicity in the comet and micronucleus assays of industrial multiwalled carbon nanotubes Graphistrength© C100 after a 90-day nose-only inhalation exposure of rats 
Graphistrength© C100 multiwalled carbon nanotubes (MWCNT) provide superior electrical and mechanical properties for various applications. The evaluation of the intrinsic hazard properties of Graphistrength© C100 is an essential step for safe use. A general feature of multiwalled carbon nanotubes after inhalation or intratracheal exposures is the induction of an inflammatory reaction in the lungs sometimes associated with local genotoxic effects.
After investigating different parameters for the aerosol generation and performing a 5-day inhalation range finding study, male and female Wistar rats were exposed nose-only for 90 days to target concentrations of 0.05, 0.25 and 5.0 mg/m3 air of Graphistrength© C100 and sacrificed 24 h and 90 days after the last exposure. Broncho-alveolar lavage fluid (BALF) was also collected and analyzed for inflammatory parameters. Twenty-four hours post-exposure, chromosomal aberrations in the bone marrow cells were evaluated by the micronucleus test and DNA damages in the lung, kidney and liver cells by both the standard and the human 8-oxoguanine DNA N-glycosylase 1 (hOGG1)-modified comet assay. All studies were performed according to the OECD test guidelines.
An inflammatory lung reaction and the release of inflammatory factors in the BALF were observed in all rats exposed to 5.0 mg/m3, associated with changes in the differential white blood cells counts. The slight changes in BALF parameters at 0.25 mg/m3 recovered and signs of lung clearance of the MWCNT were observed. No pathological changes were observed on the pleura. Neither increase in the number of micronucleated polychromatic erythrocytes nor increase in percent DNA damage were observed at any concentration.
Lung inflammation characteristic of an overload with insoluble particles was observed after a 90-day exposure to 5.0 mg/m3 of Graphistrength© C100. Clear signs of clearance and recovery were observed at 0.25 mg/m3. No genotoxicity was detected locally in lung and distally in bone marrow, liver and kidney. Therefore, Graphistrength© C100 appears of low concern in term of local and systemic genotoxicity and a No-Observed Adverse Effect Concentration (NOAEC) of 0.25 mg/m3 (0.28 mg/m3 as actual concentration) was established for the repeated-dose toxicity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12989-015-0096-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4496819  PMID: 26156627
Multiwalled carbon nanotubes; Comet assay; Micronucleus assay; Genotoxicity; Subchronic; Inhalation; Toxicity; NOAEC
24.  SP-B and SP-C Containing New Synthetic Surfactant for Treatment of Extremely Immature Lamb Lung 
PLoS ONE  2012;7(7):e39392.
Although superiority of synthetic surfactant over animal-driven surfactant has been known, there is no synthetic surfactant commercially available at present. Many trials have been made to develop synthetic surfactant comparable in function to animal-driven surfactant. The efficacy of treatment with a new synthetic surfactant (CHF5633) containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, SP-B analog, and SP-C analog was evaluated using immature newborn lamb model and compared with animal lung tissue-based surfactant Survanta. Lambs were treated with a clinical dose of 200 mg/kg CHF5633, 100 mg/kg Survanta, or air after 15 min initial ventilation. All the lambs treated with air died of respiratory distress within 90 min of age. During a 5 h study period, Pco2 was maintained at 55 mmHg with 24 cmH2O peak inspiratory pressure for both groups. The preterm newborn lamb lung functions were dramatically improved by CHF5633 treatment. Slight, but significant superiority of CHF5633 over Survanta was demonstrated in tidal volume at 20 min and dynamic lung compliance at 20 and 300 min. The ultrastructure of CHF5633 was large with uniquely aggregated lipid particles. Increased uptake of CHF5633 by alveolar monocytes for catabolism was demonstrated by microphotograph, which might be associated with the higher treatment dose of CHF5633. The higher catabolism of CHF5633 was also suggested by the similar amount of surfactant lipid in bronchoalveolar lavage fluid (BALF) between CHF5633 and Survanta groups, despite the 2-fold higher treatment dose of CHF5633. Under the present ventilation protocol, lung inflammation was minimal for both groups, evaluated by inflammatory cell numbers in BALF and expression of IL-1β, IL-6, IL-8, and TNFα mRNA in the lung tissue. In conclusion, the new synthetic surfactant CHF5633 was effective in treating extremely immature newborn lambs with surfactant deficiency during the 5 h study period.
PMCID: PMC3396642  PMID: 22808033
25.  Lung Injury after In Vivo Reperfusion 
Anesthesiology  2008;109(2):269-278.
Although short-term findings after lung reperfusion have been extensively reported, in vivo animal studies have not described outcome beyond the immediate time period. Therefore, the authors evaluated lung injury 27 h after reperfusion. They also investigated whether attenuation of lung injury with the A3 adenosine receptor agonist MRS3558 was sustained beyond the immediate time period.
In intact-chest, spontaneously breathing cats in which the left lower lung lobe was isolated and subjected to 2 h of ischemia and 3 h of reperfusion, MRS3558 was administered before reperfusion. Animals were killed 3 or 27 h after reperfusion.
When compared with 3 h of reperfusion, at 27 h the left lower lobe showed reduced apoptosis and no change in inflammation, but increased edema. Increased edema of the nonischemic right lung and hypoxemia were observed at 27 h after left lower lobe reperfusion. Increases in phosphorylated p38 levels were found at 3 h of reperfusion compared with control lung, with further increases at 27 h. The attenuation of injury observed with MRS3558 treatment at 3 h of reperfusion was sustained at 27 h.
Lung edema may worsen hours after the immediate postreperfusion period, even though lung apoptosis and inflammation are reduced or show no change, respectively. This was associated with further increases in phosphorylated p38 levels. The nonischemic lung may also be affected, suggesting a systemic response to reperfusion. In addition, early attenuation of injury is beneficial beyond the immediate period after reperfusion. Treatment aimed at inhibiting p38 activation, such as A3 receptor activation, should be further studied to explore its potential long-term beneficial effect.
PMCID: PMC2688693  PMID: 18648236

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